(a) Field of the Invention
The invention relates to a method for mapping and isolating regions of chromosomes that interact together or associate functionally in vivo. The method of the present invention is also referred to as recombination access mapping (RAM).
(b) Description of Prior Art
Currently in the field of molecular biology, it is becoming quite evident that gene regulation occurs through a complex network of processes. Transcription, replication and recombination of DNA must occur in a timely and appropriate manner or the outcome may be disastrous. Extreme control over these processes is required during development and differentiation of tissues in multicellular organisms. In contrast, disorder of these processes occurs during oncogenesis. Cancer cells often exhibit aberrant expression of genes as well as general genomic instability, a hallmark of which is an increase in recombination rates.
Activation or repression of gene expression by transcription factors or repressors respectively has been studied in great detail both in vitro and in vivo. Changes in chromatin structure are intimately linked to the activation or inactivation of a gene and can affect the replication or recombination of DNA. Mostly in vitro studies and limited in vivo data has been used to determine such changes in chromatin as they relate to DNA transcription, replication and recombination. From this data it is apparent that chromatin is organized in DNA loop domains that are organized through interaction with a nuclear protein matrix. These domains average in size from 60-100 kb and are assumed to be flanked by matrix attachment regions (MARS). MARS have been associated with functional domains of transcription and replication. Indirect methods such as DnaseI sensitivity and DNA cleavage assays involving topoisomerase inhibitors, have provided further evidence of higher order chromatin domains which may correspond to single loops (60-100 kb) and loop arrays (300 kb). Functional chromatin domains seem to exist for transcription and replication (reviewed by in Jackson, D. A., Bioessays 17:587-591, 1995). Strong evidence exists which suggests chromatin structure also plays a role in recombination as in VDJ recombination, formation and repair of double strand breaks in irradiated cells, and during meiosis; differences in meiotic recombination between imprinted domains.
Therefore, the central question is raised as to the interaction between chromatin domains and their accessibility to biological molecules are involved in gene regulation. Many studies of chromatin focus on in vitro data at the nucleosomal level (Wolffe, A. Chromatin: Structure and Function, Second Ed. Academic Press Inc. San Diego 1995). Unfortunately, it is most likely that large scale changes in chromatin packaging beyond the nucleosomal level are primarily responsible for the maintenance of certain chromatin states, such as early or late replication and hetero vs. euchromatin. Very few techniques exist to analyze DNA interaction in vivo in a global fashion over the entire genome.
Ectopic gene targeting is an alternative outcome of the gene targeting process in which a targeting vector acquires sequences from a genomic target but proceeds to integrate elsewhere in the genome. More specifically, ectopic gene targeting is a process by which an extra chromosomal molecule (recipient) obtains DNA sequence from a target locus via one-end invasion and gene conversion followed by release of the recipient molecule and integration, complete with the newly acquired sequence from the target locus, Aelsewhere in the genome. Such events were first observed in gene targeting experiments involving the adenine phosphoribosyl transferase (APRT) locus in CHO cells (Adair, G. M., et al., Proc. Natl. Acad. Sci. USA 86:4574-4578, 1989) and in experiments involving retroviral transfection of rat cells (Ellis, J. et al., Mol. Cell. Biol. 9:1621-1627, 1989). Consequently, a model has been proposed for the mechanism of ectopic gene targeting (Belmaaza, A., et al., Nucl. Acids Res. 18:6385-6391, 1990; and Belmaaza, A. et al., Mut. Res. 314:199-208, 1994). Instances of ectopic gene targeting and/or ectopic gene conversion have been seen in Drosophila (roo element, p and hobo elements), plants, yeast (between dispersed repeated genes or Ty 1 repeat elements), fungi (in Ustilago maydis, chickens (Ig rearrangement)), rabbit (generation of antibody repertoire), mice (germline ectopic gene conversion in spermatids, gene conversion between Line-1 elements, and humans (gene conversion between Line-1 elements and pseudo autosomal region on X and Y chromosome).
Although the phenomenon of ectopic gene targeting is well documented, the question of where the recipient molecule integrates, with respect to the target locus, has not been determined. It is apparent from Southern analysis that the recipient integrates in most cases at a distinct site from the target but Southern analysis does not permit the determination of the relative position of the ectopic sites with regard to the target locus.
It would be highly desirable to be provided with a method allowing the identification of interactions between chromatin domains within or between chromosomes which may be involved in gene regulation. With such a method, the functional organization of the genome could be mapped. The understanding the three-dimensional (3-D) in vivo interactions between chromosome could help to better understand complex gene regulation during cancer or other disease states.
One aim of the present invention is to provide a method allowing to define functional organization of chromatin in vivo with respect to interchromosomal and interchromatin domain interactions involved in gene regulation, including but not limited to replication, transcription and recombination.
Another aim of the present invention is to provide a method allowing to mark domains of chromatin that interact functionally in vivo with a given gene locus for the purpose of cloning such domains or their visualization in 3-D using confocal fluorescent microscopy.
Another aim of the present invention is to provide a method allowing to define points of interaction between chromosomes involved in translocation or ectopic gene conversion within or between chromosomes.
Another aim of the present invention is to provide a method allowing to define chromatin domain interactions between chromosomes involved in epigenetic phenomenon such as imprinting, position effect variegation and transvection.
Another aim of the present invention is to provide a method allowing to produce diagnostic ectopic gene targeting distribution profiles, such as fingerprints, for a given gene locus.
Another aim of the present invention is to provide a method allowing to determine changes in genomic organization associated with various disease states as a means of monitoring disease progression or onset.
Another aim of the present invention is to provide a method allowing to study developmental changes in multicellular organisms such as during tissue development.
Another aim of the present invention is to provide a method allowing for the placement of DNA elements or recognition sites for enzymes for the purpose of chromosomal engineering.
Another aim of the present invention is to provide a means for mapping the distribution of double strand breaks in DNA, which are natural breaks or induced by any means, over a given region of a chromosome with respect to a chromosomal DNA sequence to be studied, which in turn allows for the definition, characterization and cloning of structural and/or functional genomic domain(s) containing the chromosomal sequence being studied.
A further aim of the present invention is to provide a method allowing to assess the affects of a given drug or chemical on genomic organization and stability such as for defining oncogenic potential of a substance.
A still further aim of the present invention is to provide a method allowing to define at what time in a cell cycle chromatin domains associate functionally.
In accordance with the present invention there is provided a method hereinafter referred to as recombination access mapping (RAM), that enables the elucidation of DNA interaction between domains of chromatin within the genome, in vivo.
In accordance with the present invention, there is provided a method for identifying chromosomal regions having a physical proximity within a living cell, comprising the steps of:
a) providing a linear DNA vector having a first end and a second end, the first end of the vector comprising a nucleotide sequence homologous to a first region of a chromosome and being capable of homologous recombination thereto, the first chromosomal region comprising a known non-repetitive DNA sequence; the second end of the vector comprising a nucleotide sequence non-homologous to the chromosomes of the cell and being capable of illegitimate integration with a second region of a chromosome in physical proximity to the first chromosomal region;
b) introducing the DNA vector into the cell;
c) allowing the first end of the vector to recombine with the first chromosomal region, and the second end to illegitimately integrate with the second chromosomal region; and
d) detecting the homologous recombination event and the second chromosomal region whereupon the second end of the vector illegitimately integrated, thereby identifying chromosomal regions having a physical proximity within said living cell.
In accordance with another embodiment of the present invention, there is provided a method for locating in chromosomes of a living cell a DNA double strand break having a physical proximity with a known non-repetitive DNA sequence, comprising the steps of:
a) providing a linear DNA vector having a first end and a second end, the first end of the vector comprising a nucleotide sequence homologous to a first region of a chromosome comprising a known non-repetitive DNA sequence and being capable of homologous recombination thereto, the second end of the vector comprising a nucleotide sequence non-homologous to the chromosomes of the cell and being capable of illegitimate integration into a DNA double strand break of a chromosome in physical proximity to the first chromosomal region;
b) introducing the DNA vector into the cell;
c) allowing the first end of said vector to recombine with the first chromosomal region and the second end to illegitimately integrate into the DNA double strand break; and
d) detecting the homologous recombination event and the DNA double strand break whereupon the second end of the vector illegitimately integrated, thereby locating the DNA double strand break having a physical proximity with said known non-repetitive DNA sequence.
In accordance with the present invention, there is also provided a linear DNA vector having a first end and a second end. The first end of the vector comprises a nucleotide sequence capable of homologous recombination to a first region of a cell chromosome comprising a known non-repetitive DNA sequence. The second end of the vector comprises a nucleotide sequence non-homologous to the chromosomes of the cell and is capable of illegitimate integration with a second region of a chromosome, the second chromosomal region being in physical proximity to the first chromosomal region.
The DNA vector of the invention can be used for identifying chromosomal regions having a physical proximity within a living cell, and/or for locating in chromosomes of a living cell a DNA double strand break having a physical proximity with a known non-repetitive DNA sequence.
Preferably, the DNA vector of the invention further comprises, at each one of its ends, at least one detection element selected from the group consisting of a tag, a label, a signal, a polymerase chain reaction (PCR) primer sequence, a reporter gene and a portion of a reporter gene.
More preferably, the DNA vector comprises at least one signal selected from the group consisting of fluorescence in situ hybridization signals (FISH), radioactive in situ hybridization signals, confocal microscopy in situ hybridization signals and electron microscopy in situ hybridization signals. The signal(s) allows for detection of in situ hybridization of the DNA vector.
According to the present invention, the first region of a chromosome may be modified to introduce a first part of a reporter gene, and wherein the linear DNA vector comprises a second part of the reporter gene, such that recombination of the first end of the vector with the first chromosomal region, and illegitimate integration of the second end with the second chromosomal region allows the reporter gene to be functional. The reporter gene may be a selection gene selected from the group consisting of neomycin, puromycin, hygromycin, and herpes simplex thymidine kinase.
The first end of the linear DNA vector according to the invention may have a nucleotide sequence of at least 300 bp in length, preferably of at least 500 pb in length, and more preferably of about 700 pb in length. The second end of the vector is at least 1 Kb in length, preferably of about 10 Kb in length.
The method of the present invention as described above may preferably be used to characterize or identify functional or structural sequence elements such as origins of replication, matrix attachment sites, transcription factor binding sites, imprinting centers or insulator elements. Of course, the double strand breaks may be have been formed in vivo or in vitro.